Use of a fertility gene ms2 of rice

Abstract

The application discloses application of a rice fertility gene MS2. The application finds a gene MS2 for regulating rice fertility in a rice genome database, the gene is highly expressed during anther development period of rice, nucleotide sequences of the gene are shown in SEQ ID NO. 1, and the amino acid sequences are shown in SEQ ID NO. 2. Research shows that the MS2 gene is related to rice fertility, and influences rice pollen fertility, and knocking out the MS2 gene in wild type rice can make the rice pollen fertility significantly decrease, and the MS2 gene can be applied to creation of a rice sterile line and preparation of hybrid seeds. Meanwhile, the male sterile line generated by mutation of the MS2 gene is not influenced by environmental conditions such as illumination and temperature, the fertility of the male sterile line is stable, a new gene resource is provided for construction of a rice hybrid breeding system, and germplasm resources of the male sterile line are also expanded.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular breeding and genetic engineering, and more specifically relates to an application of a rice fertility gene MS2. Background Art

[0002] Rice (Oryza sativa L.) is one of the world's three major staple crops, providing food for nearly 50% of the global population. Rice is my country's primary staple food crop, accounting for one-third of the annual grain-sown area. Therefore, rice production is directly related to our country's food security. The "Green Revolution" of dwarf rice breeding in the 1960s and the "Second Green Revolution" of hybrid breeding significantly increased rice yields. Hybrid rice, with its genetic diversity and complementarity, offers advantages not only in yield but also in stress and disease resistance compared to conventional varieties. However, hybrid rice breeding faces challenges such as high seed production costs and complex technical requirements, particularly the lack of germplasm resources for male sterile lines.

[0003] The selection and breeding of male sterile lines is a key step in hybrid seed production. Male sterility diversity is an effective way to ensure hybrid rice germplasm diversity and mitigate production risks associated with germplasm homogeneity. Currently, male sterility can be categorized into cytoplasmic male sterility (CMS) and genic male sterility (GMS), depending on the gene source and mode of inheritance. CMS is widely found in higher plants and is generally caused by mitochondrial gene mutations leading to loss of function or abnormalities. This manifests as maternal inheritance, normal pistil function, but pollen abortion. In most cases, male sterility can be restored by the use of nuclear-encoded fertility restorer genes (Rf). Unlike CMS caused by cytoplasmic sources (usually mitochondrial gene abnormalities), male sterility caused by nuclear genetic abnormalities is termed GMS. Sterile lines constructed using recessive genic male sterility genes relieve environmental constraints on hybrid breeding, eliminate potential production risks, and are applicable to most varieties. They can significantly improve the utilization of heterotic resources and address the resource utilization issues associated with heterotic vigor.

[0004] Since there is still a lack of genetic resources that can be used to construct male sterile lines, it is necessary to develop more genes related to rice fertility, which is of great significance to the creation of rice sterile lines. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortage of existing gene resources for constructing male sterile lines and provide an application of rice fertility gene MS2.

[0006] The purpose of the present invention is to provide an application of rice MS2 gene.

[0007] Another object of the present invention is to provide the use of a reagent for knocking out or inhibiting the expression of the rice MS2 gene.

[0008] Another object of the present invention is to provide a method for changing rice breeding properties or cultivating / creating rice male sterile lines.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention discovered a gene that regulates rice fertility in the rice genome database, and obtained the MS2 (MALE STERILITY 2) gene that is highly expressed during the rice anther development period through bioinformatics analysis and screening. This gene is not a light-temperature sensitive gene. Its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2. Studies have shown that the MS2 gene is related to rice fertility and affects rice pollen fertility. Knocking out this gene in wild-type rice can significantly reduce rice pollen fertility, and can be better applied to the creation of rice sterile lines and the preparation of hybrid seeds. The male sterile line produced by the MS2 gene mutation provided by the present invention has stable fertility and is not affected by environmental conditions. It can help produce rice sterile lines that are not affected by light and temperature, and provides new gene resources for constructing more rice hybrid breeding systems. It also expands the germplasm resources of male sterile lines.

[0011] Therefore, the present invention provides the use of rice MS2 gene in negatively regulating rice fertility.

[0012] The present invention provides application of rice MS2 gene in reducing pollen fertility.

[0013] The present invention provides an application of rice MS2 gene as a target in cultivating / creating rice male sterile plants, specifically knocking out the MS2 gene in rice.

[0014] The present invention provides application of a reagent for knocking out or inhibiting rice MS2 gene expression in reducing rice anther fertility.

[0015] The present invention provides use of a reagent for knocking out or inhibiting rice MS2 gene expression in preparing a product for reducing rice anther fertility.

[0016] The present invention provides the use of a reagent for knocking out or inhibiting rice MS2 gene expression in cultivating / creating rice male sterile plants.

[0017] Preferably, the reagent is a plasmid, vector, or recombinant bacterium containing a gene for knocking out or inhibiting the expression of rice MS2 gene.

[0018] More preferably, the reagent is a rice MS2 gene CRISPR / Cas9 knockout vector, or a recombinant bacterium containing the knockout vector.

[0019] Furthermore, the primer set used to construct the rice MS2 gene CRISPR / Cas9 knockout vector includes Primer1 (SEQ ID NO.3) / Primer2 (SEQ ID NO.4), Primer3 (SEQ ID NO.5) / Primer4 (SEQ ID NO.6).

[0020] The present invention provides a method for changing rice breeding or cultivating / creating rice male sterile lines, knocking out or inhibiting the expression of rice fertility gene MS2 to obtain plants with reduced fertility.

[0021] Preferably, gene editing technology is used to knock out and mutate the rice fertility gene MS2 to obtain plants with reduced fertility.

[0022] More preferably, a recombinant gene CRISPR / Cas9 knockout vector containing the MS2 gene target sequence is constructed and transformed into wild-type rice to obtain plants with reduced fertility.

[0023] Further preferably, the method for constructing the CRISPR / Cas9 knockout vector is: inserting the target fragment of the gene MS2 into the BsaI recombination site of the pYLCRISPR / Cas9Pubi-H vector to obtain a recombinant plasmid.

[0024] The present invention has the following beneficial effects:

[0025] The present invention discovered a gene that regulates rice fertility in the rice genome database. Through bioinformatics analysis and screening, the MS2 (MALE STERILITY 2) gene was found to be highly expressed during the rice anther development period. This gene is not a light-temperature sensitive gene. Its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2. Studies have shown that the MS2 gene is related to rice fertility and can affect rice pollen fertility. Knocking out this gene in wild-type rice can significantly reduce rice pollen fertility, which can be better applied to the creation of rice sterile lines and the preparation of hybrid seeds. At the same time, the decrease in pollen fertility caused by the MS2 gene mutation is not affected by environmental conditions such as light and temperature, providing new gene resources for the construction of more rice hybrid breeding systems, and also expanding the germplasm resources of male sterile lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 (a) The expression pattern of MS2 candidate genes and (b) the quantitative analysis results at different stages of anther development.

[0027] Figure 2 Heat map of male fertility-related genes screened for expression analysis based on public databases.

[0028] Figure 3 Design of two CRISPR / Cas9 knockout targets in MS2 gene exons and sequencing results of target editing.

[0029] Figure 4 The plant morphology of the wild-type rice variety Tianfeng B (WT) and the ms2-ko mutant at the heading stage.

[0030] Figure 5 These are the anther morphological observation pictures of wild-type Tianfeng B (WT) and ms2-ko mutant.

[0031] Figure 6 The results of iodine staining analysis of pollen of wild-type Tianfeng B (WT) and ms2-ko mutant are shown.

[0032] Specific implementation party

[0033] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0035] Example 1 Acquisition of Rice Fertility-Related Genes and Construction of Knockout Vectors

[0036] 1. Acquisition of rice genes

[0037] First, the RiceXPro rice database (https: / / ricexpro.dna.affrc.go.jp / ) was used to search and screen genes specifically expressed in rice anthers, such as Figure 1 Then the collected genes were clustered and analyzed based on gene expression and a heat map was drawn, as shown in a. Figure 2 As shown, a gene with a high expression level during the anther development period of rice was screened out. It is not a light-temperature sensitive gene and is named MS2 (MALE STERILITY 2). The nucleic acid sequence of the MS2 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0038] 2. Quantitative analysis

[0039] Based on the nucleic acid sequence of the MS2 gene, primer pairs Primer5 (SEQ ID NO.7): 5'-GAATCTGGCGCTCCACCCCT-3', Primer6 (SEQ ID NO.8): 5'-ACAGCGTCCCTGATGCTGC-3' were designed and synthesized, and quantitative analysis of the selected target genes was performed. cDNA of rice samples from different stages (SC: sporulation stage; MMC: sporocyst stage; Meiosis: meiosis stage; EM: early uninucleate stage; LM: late uninucleate stage; BCP: dinucleate stage) was diluted 10 times and used as template to determine the expression of the target gene. The rice gene OsUFC1 was selected as the internal reference. Three parallel control experiments were set up for each sample group each time. The specific expression of the target gene was analyzed using the relative quantitative method; the relative value of the target gene to UFC1 was calculated using the expression of UFC1 as a reference. The calculation method was 2 -△Ct The relative expression value was calculated as 2 -△Ct (2- [Cq目的-Cq内参] ).

[0040] The RT-qPCR amplification system was as follows: 10 μL 2× ChamQ Universal SYBR qPCR MasterMix, 0.5 μL 10 μM Forward Primer, 0.5 μL 10 μM Reverse Primer, 1 μL template cDNA, and ddH₂O to a total of 20 μL. The reaction procedure was: 1 cycle at 94°C for 10 min (pre-denaturation); 40 cycles at 94°C for 10 s (denaturation), 60°C for 15 s (annealing), and 72°C for 15 s (extension). The melting curve temperature range was set to gradually increase from 65°C to 94°C, and the fluorescence signal was recorded every 0.5°C increase.

[0041] Quantitative test results such as Figure 1 As shown in b, the MS2 gene is expressed in multiple stages of anther development, with the highest expression level in the sporocyst stage.

[0042] 3. Construction of knockout vector

[0043] Double target knockout was performed on two sites T1 (SEQ ID NO.9): 5'-GATGGACACGATCCTCCACTTGG-3', and T2 (SEQ ID NO.10): 5'-CCTCATCGACGGCGACCTCTTCC-3' on the 2nd and 6th exons of the MS2 gene, such as Figure 3 As shown, the two targets have a common feature, with NGG (N is any base of A, T, C, or G) at the 3' end.

[0044] Two pairs of primers were designed and synthesized for these two target sites: Primer1 (SEQ ID NO.3): 5'-ATGGACACGATCCTCCACTgtttcagagctagaaat-3', Primer2 (SEQ ID NO.4): 5'-AGTGGAGGATCGTGTCCATCggcagccaagccagca-3'; Primer3 (SEQ ID NO.5): 5'-GAAGAGGTCGCCGTCGATGgtttcagagctagaaat-3', Primer4 (SEQ ID NO.6): 5'-CATCGACGGCGACCTCTTCCggcagccaagccagca-3'.

[0045] The CRISPR / Cas9 vector pYLCRISPR / Cas9Pubi-MS2 containing the above two targets was constructed according to the method for constructing the pYLCRISPR / Cas9Pubi-H vector (Ma X, Zhang Q, Zhu Q, et al. Arobust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant, 2015, 8(8): 1274-1284). The recombinant plasmid pYLCRISPR / Cas9Pubi-MS2 was transformed into the Agrobacterium EHA105 strain by electroporation to obtain a recombinant strain. The plasmid was extracted and identified by PCR and enzyme digestion. The recombinant strain that was correctly identified by PCR and enzyme digestion was named pYLCRISPR / Cas9Pubi-MS2.

[0046] Using the genetic transformation method of Agrobacterium-infected rice embryos (Nishimura A, Aichi I, Matsuoka M. A protocol for Agrobacterium-mediated transformation in rice. Nature Protocols. 2006, 1(6): 2796-2802), pYLCRISPR / Cas9Pubi-MS2 was transformed into the indica rice variety Tianfeng B (TFB; derived from germplasm preserved in our laboratory) to obtain transgenic T0 generation plants with MS2 gene knockout.

[0047] Example 2 Identification and decoding analysis of editing sites in transgenic plants

[0048] The T0 generation plant genomic DNA obtained in Example 1 was extracted using the SDS microgenomic DNA extraction method. Using the genomic DNA as a template, the upstream detection primer Primer7 (SEQ ID NO. 11): 5'-CTGACGCTAACCTCGACAAG-3' was designed on the CDS region of the Cas9 protein on pYLCRISPR / Cas9Pubi-MS2, and the downstream detection primer Primer8 (SEQ ID NO. 12): 5'-CCGATCTAGTAACATAGATGACACC-3' was designed on the NOS terminator. PCR amplification was then performed using Primer7 and Primer8 primers to detect whether the T0 generation plants carried the transgene.

[0049] PCR reaction system: 1 μL DNA (20 ng / μL), 0.4 μL Primer 7 (10 pmol / μL), 0.4 μL Primer 8 (10 pmol / μL), 10 μL 2× Buffer, 0.4 μL dNTPs (10 mM), 0.4 μL rTaq (5 U / μL), 0.4 μL, and ddH2O to 20 μL. PCR amplification program: 94°C for 3 min; 30 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 1 min; 72°C for 5 min.

[0050] The test results showed that all T0 generation plants carrying transgenic markers were successfully edited.

[0051] Subsequently, primers were designed upstream and downstream of the two target positions of the genomic sequence corresponding to the MS2 gene, respectively. The upstream primer Primer9 (SEQ ID NO.13) 5'-CCATGATAGCCAAACCCTAG-3' and the downstream primer Primer10 (SEQ ID NO.14) 5'-GGATGAGGAGGAAGTGAAGG-3' for the first target, and the upstream primer Primer11 (SEQ ID NO.15) 5'-GAGGATCGCATCAGCGCCCT-3' and the downstream primer Primer12 (SEQ ID NO.16) 5'-CGACAGCGTCCCTGATGCT-3' for the second target.

[0052] Amplification was performed using genomic DNA from T0 plants as a template. The PCR reaction system consisted of 1 μL DNA (20 ng / μL), 0.4 μL upstream primer (10 pmol / μL), 0.4 μL downstream primer (10 pmol / μL), 10 μL 2× Buffer, 0.4 μL dNTPs (10 mM), 0.4 μL rTaq (5 U / μL), and ddH2O to a final volume of 20 μL. The PCR amplification protocol was as follows: 94°C for 3 min, followed by 30 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 30 sec, and finally 72°C for 5 min. After amplification, Sanger sequencing was performed to detect mutations at different target sites.

[0053] Then, the sequencing peak graph was decoded using the CRISPR-GE online tool (Liu, Weizhi, Xianrong Xie, Xingliang Ma, Jun Li, Jie-hu Chen and Yaoguang Liu. “DSDecode: A Web-Based Tool for Decoding of Sequencing Chromatograms for Genotyping of Targeted Mutations.” Molecular plant, 2015, 8(9): 1431-1433.; Xie X, Ma X, Zhu Q, et al. CRISPR-GE: A convenient software toolkit for CRISPR-based genome editing. Molecular Plant, 2017, 10(9): 1246-1249.). The sequencing results are as follows: Figure 3 As shown, frameshift mutations caused by deletion / insertion were found in different target sites, and a transgenic strain with MS2 gene knockout was successfully constructed.

[0054] Example 3 Phenotypic analysis of transgenic plants

[0055] The transgenic T0 plants with MS2 gene knockout and the wild type Tianfeng B were planted at the Rice Experimental Station of South China Agricultural University. The water and fertilizer conditions were the same during the planting period, and the pollen of the plants was stained with I2-KI after flowering.

[0056] The results showed that there was no significant difference in plant type between the transgenic plants of pYLCRISPR / Cas9Pubi-MS2 and the wild-type plants at the heading stage. Figure 4 There is no obvious difference in anther morphology between different plants, such as Figure 5 However, the pollen vigor of the transgenic plants was reduced, and the pollen fertility decreased, as shown in Figure 6As shown, knocking out the MS2 gene significantly reduces rice anther fertility and can be used to create rice sterile lines. Because the MS2 gene is not a light- and temperature-sensitive gene, the sterile lines constructed by MS2 gene mutations have stable fertility and are not affected by the environment. Knocking out the MS2 gene can be used to create rice male sterile lines, helping to generate more rice sterile lines that are not affected by light and temperature, and providing more methods for constructing sterile lines using recessive nuclear sterile genes.

[0057] In summary, the present invention has discovered a gene that regulates rice fertility. Bioinformatics analysis identified the MS2 gene, which is highly expressed during rice anther development. Knockout of this gene significantly reduces anther fertility, potentially enabling the development of rice sterile lines and hybrid seed production. Furthermore, the reduced pollen fertility of sterile lines created by mutation of the MS2 gene could facilitate the development of more male sterile lines, providing a new genetic resource for rice hybrid breeding.

[0058] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Rice MS2 The application of a gene in reducing male fertility in rice is characterized in that: Knockout in rice MS2 gene; MS2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Rice MS2 The use of a gene in reducing pollen fertility is characterized in that: Knockout in rice MS2 gene; MS2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. Rice MS2 The application of a gene as a target in the cultivation / creation of rice male sterile plants is characterized in that: Knockout in rice MS2 gene; MS2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. Knockout Rice M The use of an agent for the S2 gene in reducing the fertility of rice anthers is characterized in that: described MS2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. Knockout Rice MS2 The invention relates to a method for preparing a product for reducing the fertility of rice anthers by using a gene reagent, characterized in that: described MS2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. Knockout Rice MS2 The use of a gene reagent in cultivating / creating rice male sterile plants is characterized in that: described MS2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. The use according to any one of claims 4 to 6, characterized in that: The reagent is a knockout rice MS2 Plasmids, vectors, or recombinant bacteria for gene expression.

8. A method for changing rice breeding properties or cultivating / creating rice male sterile lines, characterized in that: Knockout of rice fertility genes MS2 The rice fertility gene is expressed to obtain plants with reduced fertility; MS2 The nucleotide sequence is shown in SEQ ID NO.

1.

9. The method according to claim 8, characterized in that Using gene editing technology, the rice fertility gene MS2 Knockout mutations were performed to obtain plants with reduced fertility.

10. The method according to claim 9, characterized in that: Constructing a rice fertility gene MS2 The recombinant gene CRISPR / Cas9 knockout vector of the target sequence was used to transform wild-type rice to obtain plants with reduced fertility.

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